CENTER FOR TRANSLATIONAL RESEARCH IN ONCOLOGY
INSTITUTO DO CÂNCER DO ESTADO DE SÃO PAULO
INSTITUTO DO CÂNCER DO ESTADO DE SÃO PAULO
RESEARCH PROGRAMS
CLINICAL
RESEARCH
MOLECULAR
ONCOLOGY
Drug targeted
therapies
Molecular
Epidemiology
Pharmacoeconomics
Biomarkers and their
clinical application
Incorporation of New
Technologies at SUS
Molecular Imaging
Systems Oncology
INNOVATIVE
THERAPEUTICS
EPIDEMIOLOGY
PREVENTION
Chemosensitization
Cohort studies
Gene Therapy /
Immunotherapy
Vaccination protocols
Primary and
Secondary
Prevention
CORE FACILITIES AT UNIVERSIDADE DE SÃO PAULO
ICESP
FMUSP-HC
IN OTHER CAMPI, E.G.
Biobank
Recombinant DNA and expression vector construction
Sequencing and transcriptome profiling
Cytometry and expansion of human cancer cells
Epidemiology
Unit for Immunology (InCOR/FMUSP)
Center for Nuclear Medicine/ Molecular Imaging
Center for Transgenic Animals
NUCEL/NETCEM, Cellular and Molecular Therapy
INCTC- FMRP
INSTITUTO DO CÂNCER DO ESTADO DE SÃO PAULO
VISION: ICESP will interface the Secretary of Health and the Academic Network for
Cancer Research, allowing for the completion of translational projects of interest to
Public Health in the area of Oncology
Secretary of Health
Secretary of Health
ICESP
ICESP
Academic Cancer
Research Network
Cancer Management
Network
The tumor microenvironment as a target
for experimental combination therapy in tumors
Universidade de São Paulo, ICESP/FM
Andréia Hanada Otake
Angélica Patiño
Camila Maria Longo Machado
Camila Morais Melo
Fabiana Henriques Machado de Melo
Luciana Nogueira de Souza Andrade
Patricia Costa
Rafael Yamashita Ikemori
Verônica Rodrigues Teixeira
Emerson Soares Bernardes
Sofia Nascimento dos Santos
Roberto Zatz and group
University of Toronto
University of California, Davis
Ian Tannock
Daniel K. Hsu
Fu-Tong Liu
Ludwig Inst for Cancer Res, S Paulo
Moffitt Research Center, Tampa
Anamaria Aranha Camargo
Robert J. Gillies
Support: FAPESP, CNPq, NIGMS/NIH Consortium for Functional Glycomics and UICC-YY grant
Galectins, a family of b-galactoside binding lectins,
have a conserved carbohydrate recognition domain (CRD)
Proto
Chimera
Tandem
Repeat
CRD
CRD
CRD
CRD
Galectins 1,2,5,7,
10,11,13,14, 15
Galectin-3
Galectins 4,6,8,
9 and 12
Structures of Galectins
Crystal Structure (1.7 Å) of Dimeric Human
Galectin-1 With Bound Lactose
COONH2
NH2
COO-
Sideview
Turned 90˚
Galectins have a highly conserved secondary structure with internally oriented
hydrophobic residues in ß strands in the ß-sandwich of the galectin fold .
Prototypical galectins are found as dimers in the extracellular space, galectin-3
is found as a pentamer/aggregates in the extracellular space
Galectin-3 amplifies the intensity of macrophage-dependent immune responses
and interferes with B cell differentiation into plasma cells (Notch signalling)
M1
M2
Tumor-associated inflammation
Monocyte/
Macrophage
B cell/
Plasma cell
Infection-related granulomatous inflammation
Th1
Th2
Toxoplasma gondii
Rhodococcus equi
Schistosoma mansoni
P. brasiliensis
Bernardes et al. 2006, Am. J. Pathol. 168:1910-1920
Ferraz et al. 2006, Eur. J. immunol 38:2762-2775
Ruas et al. 2009. PLoS ONE 4:e4519
Oliveira et al. 2007, J. Leuk. Biol. 82:300-310
Oliveira et al. 2009, Glycobiology 19:1248-1258
V
Galectin-3
Neder et al.(2004) Brain Pathology 14:399-405.
P
N
BV
*
Pimonidazole-reactivity
Galectin-3
Galectin-3 expression is dynamic, responding to microenvironmental stimuli
such as hypoxia and nutrient deprivation found in glioma pseudopalisades
Ikemori et al. (2014)
Galectin-3 expression is induced and the protein accumulates in cells exposed
to hypoxia and serum deprivation
6
5
4
3
***
**
2
5.0
1
0
***
*
12 18
6
24
30
Time (h)
0
0
6
12
18
24 30
Time (h)
36
42
Normoxia
CoCl2 100uM
Hypoxia
Hypoxia
FBS
1%
13%
***
6
5
42
48
0
0
CoCl2 100uM
6
12
2
13%
1%
Gal-
3
βactin
Gal-3
relative
increase
1,0
2,55
1,0
1,39
1,86
18
24 30
Time (h)
36
42
48
Hypoxia
Hypoxic
conditions
CoCl 100M
1%
***
***
***
***
***
1 36
48
Normoxia
C Normoxia
7
4
***
*** 3
2
2.5
0.0
Gal-3 relative expression
7.5
7
1% FBS
13% FBS
Gal-3 relative expression
Gal-3 relative expression
13% FBS
Normoxia
CoCl2 100uM
Hypoxia
13%
29.7
kDa
28.8
kDa
2,15
48hs
Ikemori et al. (2014) Plos One
Upregulation of gal-3 in hypoxia and serum deprivation depends on HIF-1α
and NF-κB
Ikemori RY, Machado CML, Furuzawa KM, Nonogaki S, Osinaga E, et al. (2014) Galectin-3 Up-Regulation in Hypoxic and Nutrient
Deprived Microenvironments Promotes Cell Survival. PLoS ONE 9(11): e111592. doi:10.1371/journal.pone.0111592
http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0111592
Gal-3 knockdown sensitizes cells to cell death in oxygen and nutrient deprivation in the NG97ht
cell line.
Ikemori RY, Machado CML, Furuzawa KM, Nonogaki S, Osinaga E, et al. (2014) Galectin-3 Up-Regulation in Hypoxic and Nutrient
Deprived Microenvironments Promotes Cell Survival. PLoS ONE 9(11): e111592. doi:10.1371/journal.pone.0111592
http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0111592
U87MG glioma cells transduced with gal-3 shRNA demonstrate decreased tumor size and growth.
Ikemori RY, Machado CML, Furuzawa KM, Nonogaki S, Osinaga E, et al. (2014) Galectin-3 Up-Regulation in Hypoxic and Nutrient
Deprived Microenvironments Promotes Cell Survival. PLoS ONE 9(11): e111592. doi:10.1371/journal.pone.0111592
http://127.0.0.1:8081/plosone/article?id=info:doi/10.1371/journal.pone.0111592
Galectin-3 is present in lamellipodia of
migrating cells
Melo et al. (2011) Plos one e29313
Gal-3 null (S12) cells are more adherent and less migratory
on laminin-1 surfaces than gal-3 +/+ cells (S12)
Cell Adhesion
Cell Migration
B
50
Cell number/ field
Cell number/ filed
A
40
30
20
10
0
0.0
2.5 5.0 7.5 10.0 12.5
Laminin g/mL)
(
S12
S12
100
p<0.01
75
50
25
0
-
5 105 10
S12
S12
Laminin (g/mL)
Melo et al. (2011) Plos one e29313
Gal-3 expression in gal-3 null cells rendered them less
adherent and more migratory on laminin-1 surfaces
Cell Adhesion
C
40
30
20
10
0
P<0.01
Cell number/ field
Cell number/ field
CCR2
B
neo/gal 3
neo
A
Cell Migration
125
p<0.05 p<0.01
100
75
50
25
0
Substrate - LN LN LN
Lactose(mM)
- - 50100
- LN LN LN
- - 50 100
S12.neo S12.neo/gal 3
Melo et al. (2011) Plos one e29313
Extracellular galectin-3 destabilizes focal adhesion plaques
Melo et al. (2011) Plos one e29313
Cell migration triggered by galectin-3 is associated with AKT phosphorylati
it is dependent upon PI-3K activation
Melo et al. (2011) Plos one e2931
Increasing complexity: the role of galectin-3 in the build up of a vascularized tumor.
Tm1 melanoma cells were selected from a “normal” melanocyte cell line and
do not express galectin-3
A
-137
B
+377
Machado et al. (2014) Cancer Med. 3:201-14
Tm1 tumor growth is impaired in galectin-3 deficient mice (KOTm1 or KON3)
Galectin-3, either from tumor or stromal origin, stimulates melanoma growth
C
D
Machado et al. (2014) Cancer Med. 3:201-14
A
WTG3
KO
KOG3
G3
***
2
necrosis
Necrotic area
area/field
(µm2)(m )
100000
WTN3
KON3
WTG3 (n=8)
WTN3 (n=6)
KOG3 (n=5)
KON3 (n=7)
80000
60000
40000
Absence from galectin3 from both tumor cells
and stromal cells
(KON3) favor necrosis,
20000
CD34
10000
5000
KO
G3
TN
3
0
W
CD34
3
KON3
WTG3 (n=8)
WTN3 (n=6)
KOG3 (n=5)
KON3 (n=6)
15000
TG
associated with
decreased/unsustai
ned
angiogenesis.
CD34
*
W
WTN3
***
20000
KO
N3
KOG3
KO
N3
KO
G3
TN
CD34
2
WTG3
2) (m )
functionalvessel
vesselsarea
area(µm
/field
B
W
W
TG
3
3
0
VEGF expression is decreased within tumors devoid of galectin-3.
TGF-b accumulates in VEGF poor tumors (a compensatory mechanism?)
A
VEGF
15kDa
β-actin
40kDa
15kDa isoform
B
15kDa monomer
25kDa homodimer
Machado et al. (2014) Cancer Med. 3:201-14
Macrophage homing to tumors is altered in the absence of galectin-3.
Galectin-3 deficient macrophages produce less VEGF than wild type macrophages
(attenuated M2 phenotype of galectin-3 deficient macrophages)
KO
G3
WT
N3
KON3
WTG3
KO
G3
WT
N3
KON3
A
CD-68+ cell number (mm2)
WTG3
Machado et al. (2014) Cancer Med. 3:201-14
Davanat, a pectin that acts as a galectin inhibitor,
may be used to control tumor growth
Summary-1
Galectin-3 and “the run or die” hypothesis
Galectin-3 expression is increased in stressed tissue
microenvironments associated with tumor cell migration and
tissue remodelling, inducing angiogenesis.
Endogenous galectin-3 accumulated in tumor cells
exposed to hypoxia and nutrient deprivation. Under these
conditions, endogenous galectin-3 favoured survival (not a
lectin function).
Extracellular galectin-3 is targetable by pectins, which may
be used to disrupt tumor cell migration and tumor-associated
angiogenesis.
Imaging the tumor microenvironment in
experimental melanoma models
Angiogenesis and vascular function control as
targets for combination therapy in
experimental tumors
Vasoactive peptides and their angiogenic/vascular
permeability functions
• Angiotensin II antagonists and bradykinin antagonism
• Imag(in)ing tumor vasculature function and interfering
with tumor perfusion
Angiotensin II, Bradykinin and des-Arg-Bradykinin
control the vascular tonus and other endothelial cell functions
Angiotensin converting enzyme (ACE) connects both systems
Kininogen
Angiotensinogen
Kallikreins
B2R
Renin
Angiotensin I
Bradykinin
ACE
Carboxipeptidases
B1R
des-Arg9 -
Bradykinin
Inactive
peptides
Angiotensin II
AT1R
AT2R
Losartan, candesartan
Decrease vascular tonus
Increase vascular tonus
PRESENCE OF AT1 RECEPTORS AND
ANGIOTENSIN II IN HUMAN MELANOMA TISSUES
Otake et al.(2010) Cancer Chemother Pharmacol 66:79-87
ANGIOTENSIN II ANTAGONISTS LIMITED
MURINE MELANOMA GROWTH
Maximal tolerated dose of Losartan
Otake et al.(2010) Cancer Chemother Pharmacol 66:79-87
Low dose of losartan
Nat Commun. 2013;4:2516. doi: 10.1038/ncomms3516.
Angiotensin inhibition enhances drug delivery and potentiates chemotherapy by
decompressing tumour blood vessels.
Chauhan VP1, Martin JD, Liu H, Lacorre DA, Jain SR, Kozin SV, Stylianopoulos T, Mousa AS, Han X, Adstamongkonkul P, Popović Z,Huang P, Bawendi
MG, Boucher Y, Jain RK.
Angiotensin II, Bradykinin and des-Arg-Bradykinin
control the vascular tonus and other endothelial cell functions
Angiotensin converting enzyme (ACE) connects both systems
Kininogen
Angiotensinogen
Kallikreins
B2R
Renin
ACE
Carboxypeptidases
des-Arg9 -
B1R
Angiotensin I
Bradykinin
Bradykinin
Inactive
peptides
Angiotensin II
AT1R
AT2R
R-715, R-954
Decrease vascular tonus
Increases vascular tonus
Increase vascular permeability Induces angiogenesis
BKR1 antagonists R715 and R954 increased doxorubicin uptake within tum
CD31/Doxorubicin
PBS
R-954
...adverse effects of R954/R715 include
a transient increase in blood pressure
US2007015715-A1 Nantel et al. Derwent Innovations Index 2007-239159.
Costa el al. (2014). Cancer Lett. 345: 27-38
Chammas et al., in preparation
Chammas et al., in preparatio
Chammas et al., in preparation
Doppler studies
B16-F10 melanoma
225
ITS
HTS 3.75%
Doppler velocity (%)
200
HTS 7.5%
mannitol
175
150
125
100
0
Before
5
10
15
Minutes after intravenous injection
Injection
Chammas et al., in preparatio
Summary-2
Both angiotensin II and its receptor (AT1) are present within the tumor
microenvironment of human melanomas and murine melanomas.
The antihypertensive agent Losartan has a dual function, controlling
not only the vascular tonus, but also controlling angiogenesis.
Off label indications of old drugs (Losartan, e.g.) may help
managing cancer patients.
Opportunity for an academic clinical trial
Bradykinin receptor 1 antagonists may lead to secondary local and
transient hypertension, favouring drug delivery to experimental
tumors. Transient increase in
tumor perfusion can also be induced through usage of hypertonic
saline solutions.
Multimodality imaging allowed for devising a strategy of combination
therapy to improve drug delivery .
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Roger Chammas
[email protected]